Contribution of the Nuclear Localization Sequences of Influenza A Nucleoprotein to the Nuclear Import of the Influenza Genome in Infected Cells.

Replication of the RNA genome of influenza A virus occurs in the nucleus of infected cells. The influenza nucleoprotein (NP) associated with the viral RNA into ribonucleoprotein complexes (vRNPs) is involved in the nuclear import of the viral genome. NP has two nuclear localization sequences (NLSs), NLS1 and NLS2. Most studies have concentrated on the role of NP's NLSs using in vitro-assembled or purified vRNPs, which may differ from incoming vRNPs released in the cytoplasm during an infection. Here, we study the contribution of the NP's NLSs to the nuclear import of vRNPs in a cell culture model system for influenza infection: human lung carcinoma cells infected with viruses containing NP-carrying mutations in NLS1 or NLS2 (NLS2MT), generated by reverse genetics. We found that cells infected with these mutant viruses were defective in the nuclear import of incoming vRNPs and produced reduced amounts of newly synthesized NP, newly assembled vRNP, and progeny virus. In addition, NLS2MT-infected cells were also defective in the nucleolar accumulation of NP, confirming the nucleolar localization role of NLS2. Our findings indicate that both NLS1 and NLS2 have to be present for successful infection and demonstrate the crucial role of these two NLSs in the infection cycle of the influenza A virus.

Bioinformatics and Functional Analysis of a New Nuclear Localization Sequence of the Influenza A Virus Nucleoprotein.

Influenza viruses deliver their genome into the nucleus of infected cells for replication. This process is mediated by the viral nucleoprotein (NP), which contains two nuclear localization sequences (NLSs): NLS1 at the N-terminus and a recently identified NLS2 (212GRKTR216). Through mutagenesis and functional studies, we demonstrated that NP must have both NLSs for an efficient nuclear import. As with other NLSs, there may be variations in the basic residues of NLS2 in different strains of the virus, which may affect the nuclear import of the viral genome. Although all NLS2 variants fused to the GFP mediated nuclear import of GFP, bioinformatics showed that 98.8% of reported NP sequences contained either the wild-type sequence 212GRKTR216 or 212GRRTR216. Bioinformatics analyses used to study the presence of NLS2 variants in other viral and nuclear proteins resulted in very low hits, with only 0.4% of human nuclear proteins containing putative NLS2. From these, we studied the nucleolar protein 14 (NOP14) and found that NLS2 does not play a role in the nuclear import of this protein but in its nucleolar localization. We also discovered a functional NLS at the C-terminus of NOP14. Our findings indicate that NLS2 is a highly conserved influenza A NP sequence.

Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.